The multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) is an important modulator of neuronal function. Autophosphorylation of the enzyme converts it to a Ca2+-independent or autonomous kinase which may translocate from cytoskeleton to cytosol. The ability of CaM kinase to respond to physiological changes in intracellular free Ca2+, the dramatic effect of its Ca2+-dependent autophosphorylation on activity and localization, and its high concentration in neuronal tissue, especially in hippocampus, have led to suggestions that it is involved in synaptic plasticity. Our long range goals are to determine which signal transduction systems that elevate Ca2+ activate CaM kinase, to understand the regulation of the enzyme in vitro and in situ and to examine neuronal functions that are modulated by this kinase. We will continue our study of CaM kinase autophosphorylation in vitro using recombinant kinase produced from clones of each individual neuronal isoform of the enzyme. We will determine how phosphorylation of just 2-3 of its subunits propagates among the 12 subunits of the holoenzyme once Ca2+/calmodulin dissociates. This will be done by expressing combinations of site-directed mutants that we have and assessing whether kinase regulation and autophosphorylation are intrasubunit or intersubunit reactions. We will examine the maintenance of the modified state of the kinase in the presence of phosphatases in vitro and in the face of protein turnover in situ. Regulation of CaM kinase will be examined in PC12 and GH3 cells and in brain slices. These studies will examine whether the kinase becomes autophosphorylated and autonomous during elevation of Ca2+ in PC12 cells elicited by stimulation of the nicotinic receptor (influx of Ca2+) and the bradykinin receptor (release of intracellular Ca2+). The response of the kinase to oscillations in cellular Ca2+ will be tested. In parallel studies, a physiological function of CaM kinase, regulation of tyrosine hydroxylase, will be monitored. The entire signal transduction pathway, from stimulation of receptor and generation of the second messenger to activation of the kinase and regulation of its substrate can be followed in the same cell. In brain slice preparations active CaM kinase leading to its conversion to a Ca2+-independent form and its translocation from cytoskeleton/membrane to cytosol.